The present invention broadly relates to novel antimicrobial peptides and methods of making and using such peptides to inhibit microbial growth and in pharmaceutical compositions for treatment or prevention of infections caused by a broad range of microorganisms including gram-positive and gram-negative bacteria.
The extensive clinical use of classical antibiotics has led to the growing emergence of many medically relevant resistant strains of bacteria (1, 2). Moreover, only three new structural classes of antibiotics (the oxazolidinone, linezolid, the streptogramins and the lipopeptide-daptomycin) have been introduced into medical practice in the past 40 years. Therefore, the development of a new class of antibiotics has great significance. The cationic antimicrobial peptides could represent such a new class of antibiotics (3-5). Although the exact mode of action of the cationic antimicrobial peptides has not been established, all cationic amphipathic peptides interact with membranes and it has been proposed that the cytoplasmic membrane is the main target of some peptides, where peptide accumulation in the membrane may cause increased permeability and loss of barrier function (6, 7). Therefore, the development of resistance to these membrane active peptides is less likely because this would require substantial changes in the lipid composition of cell membranes of microorganisms.
Two major classes of the cationic antimicrobial peptides are the α-helical and the β-sheet peptides (3, 4, 8, 9). The β-sheet class includes cyclic peptides constrained in this conformation either by intramolecular disulfide bonds, e.g., defensins (10) and protegrins (11), or by an N-terminal to C-terminal covalent bond, e.g., gramicidin S (12) and tyrocidines (13). Unlike the β-sheet peptides, α-helical peptides are more linear molecules that mainly exist as disordered structures in aqueous media and become amphipathic helices upon interaction with the hydrophobic membranes, e.g., cecropins (14), magainins (15) and melittins (16). Concerning such peptides, we have explored certain factors that may be important in relation to antimicrobial activity.
The major barrier to the use of antimicrobial peptides as antibiotics is their toxicity or ability to lyse eukaryotic cells. This is perhaps not a surprising result if the target is indeed the cell membrane (3-6). To be useful as a broad-spectrum antibiotic, it is necessary to dissociate anti-eukaryotic activity from antimicrobial activity, i.e., increasing the antimicrobial activity and reducing toxicity to normal cells.
A synthetic peptide approach to examining the effect of changes, including small or incremental changes, in hydrophobicity/hydrophilicity, amphipathicity and helicity of cationic antimicrobial peptides can facilitate rapid progress in rational design of peptide antibiotics. Generally, only L-amino acids are the isomers found throughout natural peptides and proteins; D-amino acids are the isomeric forms rarely seen in natural peptides/proteins except in some bacterial cell walls. In certain circumstances, the helix-destabilizing properties of D-amino acids offer a potential systematic approach to the controlled alteration of the hydrophobicity, amphipathicity, and helicity of amphipathic α-helical model peptides (26).
Herein, we disclose the utilization of the structural framework of an amphipathic α-helical antimicrobial peptide, V681 (28), to systematically change peptide amphipathicity, hydrophobicity and helicity by single D- or L-amino acid substitutions in the center of either the polar or nonpolar faces of the amphipathic helix. Peptide V681 has been shown to have excellent antimicrobial activity and strong hemolytic activity (27, 28) and thus served as a potentially useful candidate for our study. By introducing different D- or L-amino acid substitutions, we report here that hydrophobicity/amphipathicity and helicity have dramatic effects on the biophysical and biological activities and, utilizing this method, a significant improvement in antimicrobial activity and specificity can be achieved. In addition, high peptide hydrophobicity and amphipathicity can also result in greater peptide self-association in solution. Since we have developed a useful method to measure self-association of small amphipathic molecules, namely temperature profiling in reversed-phase chromatography (29, 30), this technique was applied for the first time to investigate the influence of peptide dimerization ability on biological activities of α-helical antimicrobial peptides. The advantage of the invention will become apparent in the following description.